Rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria: the enzyme expressed in Escherichia coli acts as a member of the respiratory chain in the host cells.
The NDI1 gene encodes the internal rotenone-insensitive NADH-quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae. The T7 tag-fused mature NDI1 was overexpressed in Escherichia coli. The overexpressed NDI1 was exclusively found in the membrane fraction. The NDI1-overexpressed membranes showed significantly increased activities of NADH oxidase and NADH-ubiquinone-1 (UQ1) reductase when compared with the control membranes. Flavone, which is a specific inhibitor of the S. cerevisiae NDI1, inhibited almost completely NADH oxidase and NADH-UQ1 reductase activities of NDI1-overexpressed membranes but scarcely inhibited these activities of the control membranes. In addition, the NADH oxidase activity of the NDI1-overexpressed membranes was also inhibited by KCN as well as the control membranes. These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E. coli membranes are different from S. cerevisiae inner mitochondrial membranes in terms of quinones and lipid composition.
Kitajima-Ihara
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Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Yagi
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T
D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D047309:Flavones; D005419:Flavonoids / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010101:Oxygen Consumption; D011190:Potassium Cyanide / Q000494:pharmacology; D011808:Quinone Reductases / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004791:Enzyme Inhibitors; D047309:Flavones; D005419:Flavonoids; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D012402:Rotenone; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases; D011190:Potassium Cyanide; C043562:flavone